1. Field of the Invention
The present invention relates to a process for producing an immobilized enzyme composition. Particularly, the present invention relates to a process for producing an immobilized enzyme composition which comprises simultaneously reacting a non-proteolytic enzyme and a water-soluble protein polymer compound with non-hardened granular gelatine by means of a water-soluble multi-functional compound.
2. Description of the Prior Art
There have hitherto been various proposals for the production of an immobilized granular enzyme wherein an enzyme is bonded to a water-insoluble granular carrier.
For instance, there is a method in which glucose isomerase is bonded to a granular ion exchange resin (Yokote et al., Die Starke 27, 302 (1975)), a method wherein glucoamylase is bonded to a porous ceramics carrier (D. D. Lee et al., Die Starke 27, 384 (1975)), or a method in which aminoacylase is bonded to e.g. acrylamide in such a manner that an aqueous solution containing an enzyme or an enzyme-containing bacteria and a water-soluble monomer is polymerized and granulated (T. Mori et al., Enzymologia 43 213 (1972)).
Further, there is a method for obtaining an immobilized granular enzyme, which comprises suspending an enzyme in an aqueous solution of gelatine, adding this suspension dropwise to an organic solvent which is immiscible or hardly miscible with water, thereby to form granules, and then treating them with a multi-functional reagent (U.S. Pat. No. 3,838,007 of Sept. 24, 1974, Japanese Patent Publication No. 18794/77), a method in which an enzyme-containing solution is absorbed in a high molecular weight porous hardened protein capable of absorbing water in an amount of from 2 to 8 times the dry weight of the polymer, and then a glutaraldehyde solution is added (DP 2,636,206 of Aug. 12, 1976, Japanese Laid-Open Patent Application No. 41489/78), a method which comprises treating gelatine-coated sand particles with glutaraldehyde, mixing them with a paste comprising an enzyme and polyethylene imine and then treating them with glutaraldehyde (U.S. Pat. No. 4,266,029), or a method in which a non-proteolytic enzyme and a water-soluble multifunctional reagent are reacted with granular gelatine in an aqueous medium (Japanese Laid-Open Patent Application No. 157892/79).
Among these conventional methods, the inventions disclosed in the Japanese Patent Publication No. 18794/77 (U.S. Pat. No. 3,838,007) and the Japanese Laid-Open Patent Application No. 41489/78 (DP 2,636,206) are comparable with the present invention. In the former, an enzyme is suspended in an aqueous gelatine solution and then granulated, and in the latter, an aqueous enzyme solution is absorbed in a water absorptive hardened protein. Thus, in each case, the enzyme is excessively distributed throughout the entire inner structure of the carrier granules and immobilized. Accordingly, if such an immobilized enzyme is used for an enzymatic reaction with a substrate, the substrate and the reaction product encounter undesirable diffusion resistance in the inner structure of the carrier granules, whereby the apparent enzymatic reaction rate is decreased, resulting in a decrease of the efficiency of the enzyme. On the other hand, the reaction product tends to be locally concentrated within each granule, and thus it is likely that undesirable side reactions are thereby led. Especially when a polymer substrate is used, the enzymatic reaction rate is considerably reduced as such a substrate has a small diffusion velocity into the carrier granules.
A conventional method which is closest to the present invention is the above-mentioned Japanese Laid-Open Patent Application No. 157892/79, in which an enzyme solution and a multi-functional cross linking agent are simultaneously added to and reacted with a non-hardened water-soluble gelatine. According to this method, the gelatine starts to swell from its surface, and at the same time, the cross linking reaction proceeds from the surface. Consequently, a dense net work structure is formed on its surface before the enzyme diffuses into the inside of the gelatine granules. Accordingly, the enzyme is hardly able to penetrate into the inside of the gelatine granules and it undergoes a cross linking reaction with the gelatine to form an insoluble enzyme locally concentrated at the gelatine surface. Thus, an immobilized enzyme composition having a high enzymatic efficiency is obtainable which is free from a decrease in the apparent reaction rate due to the diffusion resistance against the penetration of the substrate into the inside of the granules.
However, the immobilized enzyme composition obtained by this method has a drawback that when used for a long period of time, its enzymatic activity decreases. This is a serious problem when it is used as an industrial catalyst.
Namely, it has been found tha when the enzyme composition is used for a long period of time in an industrial reactor such as a fixed bed reactor, the enzyme locally concentrated at the surface of the gelatine granules splits from the gelatine granules by the breakage of the chemical bond between the enzyme and the gelatine, and the enzyme layer is thus peeled off, thereby resulting in a decrease of the enzymatic activity. This is caused by the fact that the bond between the enzyme and gelatine by means of the multi-functional cross linking agent is weak.